Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements).

Jae Woong Choi,Min Jeong Kim,Sung Sun Yim,Ki Jun Jeong

doi:10.1186/s12934-015-0401-7

Jae Woong Choi, Min Jeong Kim + Show 2 more

Open Access

https://doi.org/10.1186/s12934-015-0401-7

Copy DOI

Abstract

BackgroundIn most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain.ResultsFrom the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA).ConclusionsOur findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C. glutamicum, emphasizing the importance of developing IS element free host strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0401-7) contains supplementary material, which is available to authorized users.

Highlights

In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability
If the IS element is inserted in the green fluorescent protein (GFP) coding sequence, cells cannot produce functional GFPs, and those cells can be selectively sorted by a high–speed fluorescent activated cell sorter (FACS) sorter
In this study, we isolated the major IS elements in C. glutamicum and successfully developed two IS elementdeleted C. glutamicum strains to minimize the harmful effect of IS elements on the production of target biomolecules

Summary

Introduction

Various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain. In C. glutamicum, a few studies on the effects of MGEs on the bacterial cells and their protein productivities have been reported. There was an attempt to construct chassis strain of C. glutamicum by removing 36 unnecessary gene clusters which include some of IS elements [13]

Methods

Results

Discussion

Conclusion